Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.594785
Title: The role of CD38 expression on NAD levels and cell physiology in a leukaemia model
Author: Al-Abady, Zainab N.
ISNI:       0000 0004 5348 3106
Awarding Body: University of Plymouth
Current Institution: University of Plymouth
Date of Award: 2014
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Abstract:
CD38 is a transmembrane glycoprotein with both ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase activities; it is also known as a cell surface receptor. CD38 utilizes NAD(P) as a substrate to produce the second messengers, Nicotinic acid adenine dinucleotide phosphate (NAADP) and Cyclic adenosine diphosphate ribose (cADPR). CD38 has been implicated in several diseases. For instance, in chronic lymphocytic leukemia (CLL), it is known as a poor prognostic marker and as a disease modifier. Also, abundant data are available on the receptor functions of CD38 in CLL. However, the aim of the work described in this thesis was to investigate the enzymatic functions of CD38 in leukemia. The work also addresses the question of why CD38+ subset leukemia patients are characterised by poor outcome. It has been postulated that CD38 is the major NADase in cells, and that knocking it down increases NAD levels significantly. Thus, it was hypothesized that NAD levels might be depleted and result in detrimental consequences on cell physiology when CD38 is significantly expressed. Also, it was suggested that a similar linkage might be also present in leukemia, contributing to poor outcome. To test this hypothesis, a human leukaemia cell line (HL60) was used as a convenient model that differentiates into CD38+ cells when stimulated using all-trans retinoic acid (ATRA). It is shown that CD38 is expressed extracellularly and intracellularly in the differentiated cells, as evaluated by qPCR, FACS, Western blotting and the NGD cyclization assay. However, one of the major consequences of the early expression of CD38 (at 3 h) was a substantial depletion of intracellular NAD+ levels that was apparent by 4 h after treatment with ATRA. These novel data suggest a major role for CD38 as a main regulator of NAD during the differentiation. The main role of CD38 in degradation of NAD was confirmed by using a CD38 inhibitor (kuromanin). Interestingly, the drop in NAD+ levels during the differentiation was reversed after treatment with kuromanin. Furthermore, the CD38 homologue, CD157, and other NAD-consuming enzymes (PARP and SIRT) were all investigated, and it was found that there are no substantial roles of all these enzymes on the NAD+ degradation during the differentiation. In contrast, qPCR results for NAD-biosynthesis enzymes during the differentiation process showed a significant rise in indolamine 2,3-dioxygenase (IDO) mRNA expression, with lesser increases in nicotinamide nucleotide adenyltransferase (NMNAT) and nicotinamide phosphoribosyl transferase (NAMPT) mRNA levels. The consequences of low NAD levels on cell metabolism were also assessed; the results show a reduction in lactate production and glutathione levels with an elevation of TBARS levels. However, the NAD+:NADH ratio remained relatively constant. Moreover, the effects of low NAD levels on DNA repair and cell death were also investigated in response to DNA damage caused by UVB. Preliminary findings show that, in CD38+ cells, there is a resistance to apoptotic cell death. Additionally, CD38 expression was also investigated in leukaemia cells, and was found to be regulated in response to hypoxic environment, or the change in NAD+ levels following FK866, kuromanin and NAD+ application. Altogether, these studies raise the possibility that the impact of CD38 enzymatic function on NAD levels and the negative consequences on NAD(P)-dependent processes might play an important role in the poor prognosis in CD38+ leukemia patients.
Supervisor: Billington, Richard A. Sponsor: Ministry of Higher Education and Scientific Research, Republic of Iraq
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.594785  DOI: Not available
Keywords: NAD(P), CD38, HL60, leukeamia
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